DNA encoding porcine complement inhibitor

ABSTRACT

This invention is related to the DNA encoding the porcine complement inhibitor (pMCPcDNA), the porcine complement inhibitor expressed by the DNA, and a method for screening for the porcine complement inhibitor. pMCPcDNA can be obtained by preparing a cDNA library from porcine vascular endothelium and screening for cDNA encoding the porcine complement inhibitor. pMCPcDNA of this invention is useful for production of the porcine complement inhibitor by genetic recombination and for analysis for the promoter region of the porcine complement inhibitor.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/JP 96/01704 which has an Internationalfiling date of Jun. 19, 1996 which designated the United States ofAmerica, the entire contents of which are hereby incorporated byreference.

FIELD OF THE INVENTION

This invention provides DNA encoding a porcine complement inhibitor.More particularly, the invention provides the DNA encoding an inhibitorprotein which inhibits the porcine complement activity, the inhibitorprotein expressed by the gene, and a screening method for thecomplement-inhibitor gene.

BACKGROUND OF THE INVENTION

Recently, organ transplantation has been widely carried out in variouscountries. Development of highly effective immunosuppressants(Cyclosporin, FK506 and the like) has solved the problems of rejectionof organs transplanted from man to man, however, lack of donors hasbecome a serious problem. Such a problem has prompted studies onanimal-to-man organ transplantation, namely xenotransplantation.Although approximately 3,500 heart transplantations are being performedannually in European countries and the United States, they cover onlyapproximately 20 to 30% of patients who need heart transplantation. Useof animals closely related to human beings as donors (for example, suchprimates as baboons, chimpanzees and the like) involves a great deal ofdifficulty due to shortage of these animals and their high intelligence,but use of domestic animals as donors involves less problems.Particularly, pigs have advantages of easy supply due to mass rearing,their organ sizes similar to those of man, and established basictechnology including maintenance of the strains. Consequently, organtransplantation from pigs to man has been studied.

Of rejections occurring in pig-to-man organ transplantation, acuterejection by Major Histocompatibility Complex (MHC)-related cellularimmunity may not occur, since evolutional relatedness between pigs andman is so scarce that there is no similarity between their MHCs.Moreover, application of such effective immunosuppressants may avoidsuch rejection, if ever occurs.

Human blood, however, contains endogenous antibodies against pigs(namely, natural antibodies). Consequently, if a porcine organ istransplanted to man, the natural antibodies recognize the organ(antigen) resulting in formation of antigen-antibody complexes, whichactivate human complements. The activated human complements causenecrosis of the transplanted organ (rejection). Such a phenomenon occursimmediately (within an hour) after transplantation, so it is termedhyperacute rejection.

No drug inhibiting hyperacute rejection caused by complement activationhas ever been developed. No human organ is injured by human complements,since factors preventing complement activation are expressed in humanorgans. Such factors are named complement inhibitors (orcomplement-inhibiting factors). Of the complement inhibitors, threefactors, DAF (decay accelerating factor, CD55), MCP (membrane cofactorprotein, CD46) and CD59, are important. It is believed that DAF and MCPinhibit activation of complements by accelerating the destruction of C3band C3/C5 convertase, and CD59 does so by inhibiting the C9 step.

The complement inhibitors are species-specific. Porcine complementinhibitors can inhibit the complement activity of pigs but not that ofman. The porcine complement inhibitors cannot inhibit human complementsactivated by the porcine organ transplanted to man. Therefore,theporcine organ transplanted to human undergoes necrosis.

Such problems arising when a porcine organ is transplanted to man willbe solved, if human complement inhibitors are expressed in the porcineorgans by genetic engineering. In transplantation of the porcine heart,there will be no problem if the human complement inhibitors are beingexpressed by porcine vascular endotherial cells. From such a viewpoint,studies on recombinant pigs (transgenic pigs) integrated with humancomplement-inhibitor genes have widely been carried out.

As described above, xenotransplantation by using transgenic pigsintegrated with the human complement-inhibitor genes have been studied.Up to the present, promoters derived from the human complement-inhibitorgene and from viruses have been used to prepare such transgenic pigs.For the complement inhibitors to be expressed in pigs, however, thepromoters orginating from pigs may be more efficient. To obtain suchpromoters, cDNA of the porcine complement inhibitors is needed, however,no such cDNA has ever been known.

From these viewpoints, the present inventors conducted cloning forexpression of porcine complement-inhibitor cDNA and succeeded inobtaining cDNA which could make the cells resistant against the porcinecomplement and highly homologous to human MCP. During the course ofthese studies, the inventors found also a novel method for screening forcDNA libraries.

This invention was accomplished on the basis of such findings. Thepurposes of the invention were to provide cDNA for the porcinecomplement inhibitor, the porcine complement-inhibitor protein, and themethod for screening for the complement-inhibitor gene.

SUMMARY OF THE INVENTION

This invention relates to the base sequence defined by Sequence No. 1 orDNA comprising a part of its base sequence, particularly DNA comprisingor containing the 59th-to-1,147th bases of the DNA sequence defined bySequence No. 1.

Another invention provides the porcine complement inhibitor comprisingthe amino-acid sequence defined by Sequence No. 2; the DNA encoding theamino-acid sequence defined by Sequence No. 2 or the DNA comprising thesequence, and the method for screening for the clones possessing thecomplement-inhibitor genes by introducing a cDNA library to the hostcells, adding complements and the antibody against the host, and thenseparating the surviving hosts.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 outlines theplasmid cDNA library.

FIG. 2 shows survival rates of JY25 cells transformed with the porcinecomplement-inhibitor cDNA (pMCPcDNA) of this invention and determined bythe complement-selection method.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The base sequence of this invention defined by Sequence No. 1 is cDNAencoding the porcine complement inhibitor (hereinafter referred to aspMCPcDNA), and the cDNA is prepared, for example, by the followingmethods:

First, mRNA is prepared. mRNA can be prepared by conventional methods.Total RNA can be extracted from the cells, tissues and the likeexpressing the porcine complement inhibitors by such conventionalmethods as the guanisium-thiocyanate method, the hot phenol method andthe lithium-salt method. Poly(A)⁺RNA (mRNA) can be prepared by applyingthe RNA obtained on an oligo (dT)-cellulose column. At this step,porcine vascular endothelial cells, which are considered to be a highlyexpressing site of the complement inhibitors, are used favorably as acell source. A primary cell culture of the porcine vascular endothelialcells or established PAE cells are being favorably used (see J. Biol.Chem., 262, 4098, 1987).

Next, cDNA can be conventionally synsthesized from poly(A)⁺RNA (forexample, Gubler et al., Gene, 25, 263, 1983) or a commercially availablecDNA-synthesis kit. A cDNA library is prepared by such conventionalmethods as inserting the obtained cDNA into a phage vector λgt11 or intoappropriate plasmid vectors after separating long base pairs, ifnecessary.

pMCPcDNA is cloned from cDNA library thus prepared. Although cloning ofpMCPcDNA can conventionally be done by the plaque-hybridization method,colony-hybridization method and the method using specific conjugatedantibodies, the novel screening method utilizing the anti-complementactivity established by the present inventors is more favorably applied.

Namely, the cells possessing aimed cDNA can be cloned by introduction ofthe cDNA library into the appropriate cells, activation of thecomplement by adding both antibody against the cells (anti-serum) andcomplement components (serum of aimed species; in this case, porcineserum), and selection of the cells possessing anti-complementactivities. More particularly, as indicated in the examples describedbelow, the plasmid vector library is introduced into human lymphoblastJY25 cells (see J. Immunology, 141, 4283, 1988), and then the aimedcells are preliminarily selected by the conventional methods. Since ananti-antibiotic gene has been incorporated in the plasmid vector, thevector-introduced cells can survive and proliferate in a mediumcontaining the corresponding antibiotics. Although the cells arepreliminarily selected in an antibiotic-containing medium to increasethe selectivity, such method can be omitted sometimes.

Then, the complement activity is stimulated by addition of bothanti-JY25 antibody (antiserum) and complement components (serum of theaimed species; in this case, the porcine serum) to the preliminarilyselected cells. The cells to which the pMCP-gene-containing plasmid hasbeen introduced can survive, since the complement inhibitor is beingexpressed. By repeating selections with both the antibiotic-containingmedium and the complement, the cells containing aimed cDNA (pMCPcDNA)can be cloned.

By this method, cDNA of the porcine complement inhibitors (pMCP andother porcine complement inhibitors) can easily be obtained. Moreover,complement-inhibitor cDNA of various other animals can easily beobtained by applying this method to cDNA libraries of various animalspecies. pMCPcDNA (or cDNA fragments containing a part of them) can beisolated from the selected clones in accordance with the conventionalcDNA-isolation method. If necessary, pMCPcDNA (or its parts) can beisolated by subcloning the cDNA. If DNA is a part of pMCPcDNA, clonespossessing the full-length pMCPcDNA will be obtained by screening thecDNA library with the DNA as a probe.

Bases of pMCPcDNA thus obtained can be sequenced by such conventionalmethods as the dideoxy method or by using a sequence-determination kitcommercially available (for example, an Applied-Biosystems' DNAsequencer).

By above-described methods, cloning and sequencing pMCPcDNA wereaccomplished. pMCPcDNA obtained in the example described below consistedof 1,365 bp (Sequence No. 1). The region encoding the porcine complementinhibitor was 1,089 bp, of which approximately 600-bp translation regionwas highly homologous (70%) to human MCP cDNA. From the base sequence ofSequence No. 1, the total amino-acid residues of the porcine complementinhibitor (Sequence No. 2) was 363.

pMCPcDNA thus obtained is useful for production of the porcinecomplement inhibitor, for a sufficient amount of the inhibitor can beproduced by conventional genetic engineering with pMCPcDNA. For example,the porcine complement inhibitor can be obtained by cultivating thetransformant obtained by introducing the expression vector prepared byincorporating pMCPcDNA into a proper vector, which was then introducedinto a proper host. Such a DNA fragment encoding the amino-acid sequencedefined by Sequence No. 2 or that containing this DNA fragment may beincorporated into the vector.

Such a vector that comprises a promoter necessary for expression, SDsequence, terminator, enhancer, or various kinds of markers may be used,if necessary. As a host, such bacteria as Escherichia coli, Bacillussubtilis and the like, such microorganisms as yeast, or animal or plantcells can be used. It is commonly known by those skilled in the art thatthe aimed peptide may vary in the saccharide chain or the extent ofglycosylation depending upon the host cells used, and that the terminalamino-acid sequence of the peptide obtained is variable due tomodification of N and/or C terminal(s) of the precursor peptideexpressed in the host cells by processing with a signal peptidase andthe like.

The porcine complement inhibitor of this invention, defined by SequenceNo.2, inhibiting activation of the porcine and human complements, isuseful as a complement-activation inhibitor.

As far as possessing essentially the same effects as those of theporcine complement inhibitor of this invention, defined by SequenceNo.2, it will be validated if a part(s) of the amino-acid sequence aredeleted or replaced or inserted with other amino acids or the N and/or Cterminal(s) are conjugated with one or more amino acids, or sugarchain(s) are deleted or replaced.

As described above, by using pMCPcDNA of this invention, the porcinecomplement inhibitor can be produced by genetic engineering, andsufficient amounts of the inhibitor can be supplied. Since theamino-acid sequence of the porcine complement inhibitor can bedetermined from the base sequence of pMCPcDNA and the DNA coding theporcine complement inhibitor can be synthesized from the amino-acidsequence, the porcine complement inhibitor can also be produced bygenetic engineering with such DNA. By genetic engineering, not only theprotein consisting of a naturally-existing sequence but also those withone or more amino-acid replaces, inserts, or deletions and thoseconjugated with other proteins can be produced. Moreover, pMCPcDNA ofthis invention is useful for analysis of the porcinecomplement-inhibitor promoter region.

Complement-inhibitor cDNA of various animal species can easily beobtained by the screening method of this invention.

EXAMPLES

The present invention will be specifically explained in detail withactual examples, but the scope of the invention is not restricted tothese samples.

Example 1

1) Extraction of Total RNA From Porcine Vascular Endothelial Cells andPurification of Poly(A)⁺RNA

Primary culture cells of porcine vascular endothela or PAE cells (1×10⁸cells) were placed in 15-cm dishes and then 3 ml of a 5.5 M GTC(guanidine thiocyanate) solution was added to each dish. At this step,the mixture was viscous due to a high DNA content, so suction anddischarge of the mixture were repeated 20 to 30 times with a 30-mlsyringe with a 18-gauze needle to decrease the viscosity.

Then, after centrifugation (800 rpm), the sediment was discarded. Thesupernatant was overlaid on top of a CsTFA solution (17 ml) in apolyallomer (propylene-ethylene block copolymers) tube (40 ml) andultracentrifuged overnight. The upper GTC and intermediate DNA phaseswere carefully taken; the rest was discarded. The tube was invertedupside down to remove excess water. A 2-cm portion from the bottom wascut off and placed on ice. RNA in the tube bottom was scraped with anedge of a micro-pipette tip and dissolved in 600 μl of a 4 M GTCsolution. Insoluble substances were sedimented by light centrifugation.To the 600-μl solution, 15 μl of 1 M acetic acid and 450 μml of ethanolwere added. The solution was chilled for longer than 3 h at −20° C. andthen centrifuged for 10 min in a microtube (4° C.).

After removing the supernatant, RNA was immediately dissolved in 1 ml offreshly prepared TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). Afterlight centrifugation, the supernatant was taken. After addition of 13 μlof 2 M NaCl per 330 μl of the supernatant and then chilling for longerthan a few hours at −20° C., the total RNA was prepared.

Poly (A)⁺RNA was isolated and purified from the total RNA by an mRNApurification kit commercially available from Clontech (in principal,affinity chromatography on an oligo (dT) cellulose column). Namely,after washing several times a dT resin-packed column with a washingbuffer, a sample was loaded on the column, which was washed again withthe washing buffer. The RNA (mRNA) absorbed onto the dT resin was elutedwith an elution buffer, sedimented with ethanol, and then subjected tocDNA preparation.

2) Preparation of a cDNA Library

The mRNA (4 μg) obtained was filled up to 17 μl with distilled water,which was heated for 5 min at 68° C. After 10-min chilling on ice, thefollowing were added:

×5 First strand buffer 6 μl 0.1M DTT 3 μl RNasin 1 μl Linker primer (1.6μg/μl) 3.3 μl dNTP solution (10 mM) 2 μl

The mixture was stirred immediately after addition of them and allowedto stand for 2 min at 43° C. Super script 11 was added to it, incubatedfor an hour at 43° C., and then placed on ice.

Then, the following were added to the mixture on ice:

ddH₂O 134.3 μl ×5 Second strand buffer 48 μl dNTP solution (10 mM) 4 μl0.1M DTT 9 μl

After allowing to stand for 5 min on ice, the following were added:

DNA polymerase (5 U/μl)  12 μl Rnase H (1.5 U/μl) 2.7 μl

Immediately thereafter, the mixture was stirred, incubated for 150 minat 16° C., and 4 μl of T4 DNA polymerase (3 U/μl) was added. It was thenincubated for 15 min at 16° C. and then for 10 min at 37° C., andextracted with 250 μl of phenol/chloroform and with the same extractantcontaining 100 μl of TE. The extracts were pooled., to which thefollowing were added:

3M sodium acetate 40 μl Carrier (glycogen) 10 μl 100% ethanol   1 ml

The mixture was incubated overnight at −20° C. (or for an hour at −80°C.), centrifuged (for 20 min at 15,000 rpm), and then washed with 70%ethanol. After centrifugation (for 5 min at 15,000 rpm), the sedimentwas dissolved in 15 μl of TE.

To the solution obtained (4 μl), the following were added on ice:

10× Ligation buffer   2 μl Sal I adapter (1 μg/μl) 1.2 μl ddH₂O 9.8 μlT4 DNA ligase 10 units (approximately 3 μl)

The mixture was incubated overnight at 8° C. and then inactivated byheating for 30 min at 70° C. Then, the following were added to thesolution (20 μl):

Not I buffer (Toyobo H)   6 μl ×10 Triton   4 μl ×100 BSA 0.4 μl ddH₂O5.6 μl Not I (500 U/μl)   4 μl

The mixture was incubated for 2 h at 37° C. and then inactivated byheating for 30 min at 70° C. The following were added to the solution:

1M NaCl  7 μl ddH₂O 22 μl Glycogen (10 μg/μl)  1 μl

The mixture was loaded on a small-scale centrifuging gel-filtrationapparatus (for 3 min at 3,000 rpm), desalted with a Millipore filter(UFCP3 TK50), and then centrifuged (for 5 min at 10,000 rpm). Afterremoving the supernatant, 100 μl of TE was added to the sediment,whichwas centrifuged (for 15 min at 10,000) at −80° C. After removal of thesupernatant, and 100 μl of TE was added to the sediment, which wascentrifuged (for 15 min at 10,000 rpm). After removal of thesupernatant, 100 μl of TE (1/10) was added to the sediment, which wascentrifuged (for 20 min at 10,000 rpm). After removal of thesupernatant, the sediment was suspended in 30 μl of TE (1/10). cDNA wasobtained by centrifugation (twice each for 5 s at 5,000 rpm) by placingthe filter upside down.

Then, the following were admixed on ice:

cDNA 30 μl Expression vector [pMSF + (HygroB)] 21 μl ×10 Ligation buffer12 μl ddH₂O 56 μl T4 DNA ligase (4.6 U/μl) 1.5 μl 

After mixing, the mixture was allowed to stand overnight at 8° C.,heated for 30 min at 70° C., and desalted with a Millipore filter (UFCP3TK50). It was centrifuged (for 15 min at 10,000 rpm), and again (for 15min at 10,000 rpm) after addition of 100 μl of TE. After removal of thesupernatant, 100 μl of TE was added to the sediment, which wascentrifuged (for 15 min at 10,000 rpm). After removal of thesupernatant, 100 μl of TE (1/10) was added to the sediment, which wascentrifuged. After removing the supernatant, sediment was dispersing in30 μl of TE (1/10). This was centrifuged (for 5 s at 5,000 rpm) byplacing the filter upside down, and 30 μl of a cDNA library (1.2×10⁶clones, mean size: 1.5 kbp) was obtained. Outline of the structure ofcDNA obtained is illustrated in FIG. 1.

3) Cloning pMCPcDNA

Human lymphoblast JY25 cells at a logarithmic phase (1×10⁸/10 cuvettes)were suspended in HeBS (800 μl), to which the above-described cDNAlibrary (20 μl/10 cuvettes) was added. The mixture was electroporated(250V, 960 μF).

The electroporated cells were suspended in 200 ml of DMEM [containing20% FCS, IT (insulin/transferrin), and PS (penicillin/streptomycin)] andincubated for 24 h. The cells were resuspended in DMEM (containing 10%FCF, IT and PS) supplemented with hygromycin B in 400 μg/ml, incubatedfor 10 to 14 days (1×10⁷ cells) and then washed with PBS.

The washed cells were subjected to complement selection. Namely, DMEMfor selection (containing 10 ml of FCS+10 ml of porcine complement+80 mlof DMEM+1 ml of anti-JY25 antibody+667 μg of anti-DAF antibody+100 μg ofanti-MCP antibody) was added to the cells, which were incubated for 2 hat 37° C. After the reaction, the surviving cells were washed once withDMEM and counted by the Trypan-blue-staining method (the survivingcells: 0.1 to 1%). The anti-DAF and anti-MCP antibodies were added toimprove the selectivility. The surviving cells were washed with PBS,suspended in 100 ml of DMEM (10% FCS) and then incubated for 24 h.

Then, culture in the hygromycin-B-containing medium and the complementselection were repeated twice (the final surviving cells became 20%).

The surviving cells (1×10⁶ obtained were centrifuged (for 5 min at 1,000rpm), washed with PBS, mixed well with 400 μl of a 0.6% SDS/10 mM EDTAmixture, and then allowed to stand for 20 min at room temperature. Afteraddition of 100 μl of 5 M NaCl and mixing well (the cells discolored towhite), the suspension was allowed to stand overnight at 4° C. and thencentrifuged (for 15 min at 15,000 rpm) to obtain the supernatant. To thesupernatant, phenol/chloroform (200+200 μl) was added. Aftercentrifugation (for 2 to 3 min at 12,000 rpm), the supernatant wastaken. The supernatant, to which 10 μl (4 μg) of polyacrylamide and then1 ml of ethanol were added, was allowed to stand for 2 h at −80° C.After addition of 70% ethanol and washing, the sediment was suspended in20 μl of TE.

cDNA (2 μl) thus obtained was electroporated (2.5 kV, 25 μF, 400 Ω) into50 μl of Escherichia coli (MC1061). Immediately thereafter, 950 μl ofSOC medium was added to the sample in each cuvette (total 10 cuvettes),which was incubated for an hour at 37° C. with stirring (20 rpm). Theincubated samples were treated in the following way:

(1) The sample (15 μl/plate) was smeared on plates, which were stored at4° C.

(2) The remaining sample was added to 2 ml of TBK+, which was incubatedovernight at 37° C. with stirring, and subjected to purification inminiature scale (small-scale preparation of plasmid DNA). cDNA purifiedin miniature scale (2 ml of the sample) was suspended in 20 μl of TE. OfcDNA obtained, a 10-μl portion was digested with restriction enzymes(Xho I+Not I) and electrophoresed in agarose gel to examine for the cDNAsize. The remaining sample was electroporated into JY25 cells, whichwere selected by the complement-selection method described earlier andsubjected to viable count.

After the above-described check, 40 colonies were fished from the plateswith the highest surviving rate and incubated overnight in 2 ml of TKB+.The culture was purified in miniature scale and digested with therestriction enzymes (Xho I+Not I). After checking the size of cDNAinserted, clones were classified according to the cDNA size. cDNA havinga proper size and insertion frequency and bulk DNA (control) were eachelectroporated again into JY25 cells and analyzed by thecomplement-selection method. The results are shown in FIG. 2, whichtells that the cells introduced with the bulk cDNA [JY25(−pMCPcDNA)]showed a very low survival rate, whereas those with the selected cDNA(pMCPcDNA) [JY25(+pMCPcDNA)] showed a very high survival rate andresistance against the porcine complement.

cDNA was isolated from the cells showing a high survival rate, digestedwith the restriction enzymes (Xho I+Not I), conjugated to pBSIIKS+ andsequenced by a conventional method. The result showed that the cDNAcomprised a 1,365-bp base sequence (Sequence No. 1) and that the codingregion of the porcine complement inhibitor was 1,089 bp, of which anapproximately 600-bp translation region was highly homologous(approximately 70%) to human MCPcDNA. The amino-acid sequence of theporcine complement inhibitor from the base sequence of Sequence No.1 isshown in Sequence No. 2. The number of total amino-acid residues were363.

2 1365 base pairs nucleic acid double linear cDNA to mRNA not providedCDS 59..1147 1 GGAACTCGGA GAGGTCTCCG CTAGGCTGGT GTCGGGTTAC CTGCTCATCTTCCCGAAA 58 ATG ATG GCG TTT TGC GCG CTG CGC AAG GCA CTT CCC TGC CGT CCCGAG 106 Met Met Ala Phe Cys Ala Leu Arg Lys Ala Leu Pro Cys Arg Pro Glu1 5 10 15 AAT CCC TTT TCT TCG AGG TGC TTC GTT GAG ATT CTT TGG GTG TCGTTG 154 Asn Pro Phe Ser Ser Arg Cys Phe Val Glu Ile Leu Trp Val Ser Leu20 25 30 GCC CTA GTG TTC CTG CTT CCC ATG CCC TCA GAT GCC TGT GAT GAG CCA202 Ala Leu Val Phe Leu Leu Pro Met Pro Ser Asp Ala Cys Asp Glu Pro 3540 45 CCG AAG TTT GAA AGC ATG CGG CCC CAA TTT TTG AAT ACC ACT TAC AGA250 Pro Lys Phe Glu Ser Met Arg Pro Gln Phe Leu Asn Thr Thr Tyr Arg 5055 60 CCT GGA GAC CGT GTA GAG TAT GAA TGT CGC CCC GGG TTC CAG CCC ATG298 Pro Gly Asp Arg Val Glu Tyr Glu Cys Arg Pro Gly Phe Gln Pro Met 6570 75 80 GTT CCT GCG CTT CCC ACC TTT TCC GTC TGT CAG GAC GAT AAT ACG TGG346 Val Pro Ala Leu Pro Thr Phe Ser Val Cys Gln Asp Asp Asn Thr Trp 8590 95 TCA CCC CTC CAG GAG GCT TGT CGA CGA AAA GCC TGT TCG AAT CTA CCA394 Ser Pro Leu Gln Glu Ala Cys Arg Arg Lys Ala Cys Ser Asn Leu Pro 100105 110 GAC CCG TTA AAT GGC CAA GTT AGC TAC CCA AAT GGG GAT ATG CTG TTT442 Asp Pro Leu Asn Gly Gln Val Ser Tyr Pro Asn Gly Asp Met Leu Phe 115120 125 GGT TCA AAG GCT CAG TTT ACC TGT AAC ACT GGT TTT TAC ATA ATT GGA490 Gly Ser Lys Ala Gln Phe Thr Cys Asn Thr Gly Phe Tyr Ile Ile Gly 130135 140 GCC GAG ACT GTG TAT TGT CAG GTT TCT GGG AAT GTT ATG GCC TGG AGT538 Ala Glu Thr Val Tyr Cys Gln Val Ser Gly Asn Val Met Ala Trp Ser 145150 155 160 GAG CCC TCC CCG CTA TGT GAG AAG ATT TTG TGT AAA CCA CCT GGCGAA 586 Glu Pro Ser Pro Leu Cys Glu Lys Ile Leu Cys Lys Pro Pro Gly Glu165 170 175 ATT CCA AAT GGA AAA TAC ACC AAT AGC CAT AAG GAT GTA TTT GAATAC 634 Ile Pro Asn Gly Lys Tyr Thr Asn Ser His Lys Asp Val Phe Glu Tyr180 185 190 AAT GAA GTA GTA ACT TAC AGT TGT CTT TCT TCA ACT GGA CCG GATGAA 682 Asn Glu Val Val Thr Tyr Ser Cys Leu Ser Ser Thr Gly Pro Asp Glu195 200 205 TTT TCA CTT GTT GGA GAG AGC AGC CTT TTT TGT ATT GGG AAG GACGAG 730 Phe Ser Leu Val Gly Glu Ser Ser Leu Phe Cys Ile Gly Lys Asp Glu210 215 220 TGG AGT AGT GAC CCC CCT GAG TGT AAA GTG GTC AAA TGT CCA TATCCA 778 Trp Ser Ser Asp Pro Pro Glu Cys Lys Val Val Lys Cys Pro Tyr Pro225 230 235 240 GTA GTC CCA AAT GGA GAA ATT GTA TCA GGA TTT GGA TCA AAATTT TAC 826 Val Val Pro Asn Gly Glu Ile Val Ser Gly Phe Gly Ser Lys PheTyr 245 250 255 TAC AAA GCA GAG GTT GTA TTT AAA TGC AAT GCT GGT TTT ACCCTT CAT 874 Tyr Lys Ala Glu Val Val Phe Lys Cys Asn Ala Gly Phe Thr LeuHis 260 265 270 GGC AGA GAC ACA ATT GTC TGC GGT GCA AAC AGC ACG TGG GAGCCT GAG 922 Gly Arg Asp Thr Ile Val Cys Gly Ala Asn Ser Thr Trp Glu ProGlu 275 280 285 ATG CCC CAA TGT ATC AAA GAT TCC AAG CCT ACT GAT CCA CCTGCA ACC 970 Met Pro Gln Cys Ile Lys Asp Ser Lys Pro Thr Asp Pro Pro AlaThr 290 295 300 CCA GGA CCA AGC CAT CCA GGA CCT CCC AGT CCC AGT GAT GCATCA CCA 1018 Pro Gly Pro Ser His Pro Gly Pro Pro Ser Pro Ser Asp Ala SerPro 305 310 315 320 CCT AAA GAT GCT GAG AGT TTA GAT GGA GGA ATC ATC GCTGCA ATT GTT 1066 Pro Lys Asp Ala Glu Ser Leu Asp Gly Gly Ile Ile Ala AlaIle Val 325 330 335 GTG GGC GTC TTA GCT GCC ATT GCA GTA ATT GCT GGT GGTGTA TAC TTT 1114 Val Gly Val Leu Ala Ala Ile Ala Val Ile Ala Gly Gly ValTyr Phe 340 345 350 TTT CAT CAT AAA TAC AAC AAG AAA AGG TCG AAGTAAAACTGAT GTGCTTAAAG 1167 Phe His His Lys Tyr Asn Lys Lys Arg Ser Lys355 360 TAAAAGTTGC TGAGAGGACG TGGAATCCAG CCCCTTCCCT CTCCTGTGCTGCTGCCTGGG 1227 TCCCGTTTTG CATGTCATGA CTGTGTGCTT CCAAAAAATG CCTTTTGTTCGTATTTTTTT 1287 GCCTAAACGC ATGATTTTGT CTCTACTTGA ATTAAATCAT CACTGAATCCACGCAAAAAA 1347 AAAAAAAAAA AAAAAAAA 1365 363 amino acids amino acidlinear protein not provided 2 Met Met Ala Phe Cys Ala Leu Arg Lys AlaLeu Pro Cys Arg Pro Glu 1 5 10 15 Asn Pro Phe Ser Ser Arg Cys Phe ValGlu Ile Leu Trp Val Ser Leu 20 25 30 Ala Leu Val Phe Leu Leu Pro Met ProSer Asp Ala Cys Asp Glu Pro 35 40 45 Pro Lys Phe Glu Ser Met Arg Pro GlnPhe Leu Asn Thr Thr Tyr Arg 50 55 60 Pro Gly Asp Arg Val Glu Tyr Glu CysArg Pro Gly Phe Gln Pro Met 65 70 75 80 Val Pro Ala Leu Pro Thr Phe SerVal Cys Gln Asp Asp Asn Thr Trp 85 90 95 Ser Pro Leu Gln Glu Ala Cys ArgArg Lys Ala Cys Ser Asn Leu Pro 100 105 110 Asp Pro Leu Asn Gly Gln ValSer Tyr Pro Asn Gly Asp Met Leu Phe 115 120 125 Gly Ser Lys Ala Gln PheThr Cys Asn Thr Gly Phe Tyr Ile Ile Gly 130 135 140 Ala Glu Thr Val TyrCys Gln Val Ser Gly Asn Val Met Ala Trp Ser 145 150 155 160 Glu Pro SerPro Leu Cys Glu Lys Ile Leu Cys Lys Pro Pro Gly Glu 165 170 175 Ile ProAsn Gly Lys Tyr Thr Asn Ser His Lys Asp Val Phe Glu Tyr 180 185 190 AsnGlu Val Val Thr Tyr Ser Cys Leu Ser Ser Thr Gly Pro Asp Glu 195 200 205Phe Ser Leu Val Gly Glu Ser Ser Leu Phe Cys Ile Gly Lys Asp Glu 210 215220 Trp Ser Ser Asp Pro Pro Glu Cys Lys Val Val Lys Cys Pro Tyr Pro 225230 235 240 Val Val Pro Asn Gly Glu Ile Val Ser Gly Phe Gly Ser Lys PheTyr 245 250 255 Tyr Lys Ala Glu Val Val Phe Lys Cys Asn Ala Gly Phe ThrLeu His 260 265 270 Gly Arg Asp Thr Ile Val Cys Gly Ala Asn Ser Thr TrpGlu Pro Glu 275 280 285 Met Pro Gln Cys Ile Lys Asp Ser Lys Pro Thr AspPro Pro Ala Thr 290 295 300 Pro Gly Pro Ser His Pro Gly Pro Pro Ser ProSer Asp Ala Ser Pro 305 310 315 320 Pro Lys Asp Ala Glu Ser Leu Asp GlyGly Ile Ile Ala Ala Ile Val 325 330 335 Val Gly Val Leu Ala Ala Ile AlaVal Ile Ala Gly Gly Val Tyr Phe 340 345 350 Phe His His Lys Tyr Asn LysLys Arg Ser Lys 355 360

What is claimed is:
 1. DNA comprising the base sequence comprising SEQID NO:1 or a fragment thereof.
 2. DNA comprising the59^(th)-to-1,147^(th) bases of the base sequence comprising SEQ ID NO:1.3. A porcine complement inhibitor comprising the amino-acid sequencecomprising SEQ ID NO:2.
 4. DNA encoding the amino-acid sequencecomprising SEQ ID NO:2.
 5. A method comprising introducing a cDNAlibrary comprising DNA encoding the amino acid sequence of SEQ ID NO:2to host cells; adding anti-host antibodies and complement componentsand; selecting surviving host cells to screen for clones containing acomplement-inhibitor gene.
 6. A method comprising introducing a porcinecDNA library into host cells; adding anti-host antibodies and complementcomponents in the presence of anti-host complement inhibitor antibodies;and selecting surviving host cells to screen for clones containing acomplement inhibitor gene.